1. Field of the Invention
The present invention relates to a system for spatially selective, fixed-optics fluorescence detection in a multichannel polymeric microfluidic device, and a method for performing spatially selective, fixed-optics fluorescence detection.
2. Description of Background Art
A promising analytical tool for analyzing biomolecules such as DNA, proteins and protein complexes in a biomedical or clinical laboratory is a microfluidic device. Microfluidic devices are characterized by having one or more channels with at least one dimension less than 1 mm (typically much less than 1 mm). Common fluids used in microfluidic devices include biofluids such as whole blood samples, bacterial cell suspensions, protein or antibody solutions and various buffers. Microfluidic devices can be used for a variety of measurements including molecular diffusion coefficients, fluid viscosity, pH, chemical binding coefficients and enzyme reaction kinetics. Other applications for microfluidic devices include capillary electrophoresis, isoelectric focusing, immunoassays, flow cytometry, injection of protein samples for analysis via mass spectrometry, DNA analysis, cell manipulation, cell separation, cell patterning and chemical gradient formation. Many of these applications have utility for clinical diagnostics.
The use of microfluidic devices to conduct biomedical research and create clinically useful technologies has a number of significant advantages. First, because the volume of fluids within these channels is very small, generally sub-microliter, the amount of reagents and analytes used is quite small. This is especially significant for expensive reagents or samples.
Microfluidic devices can be fabricated using processes developed for the microelectronics industry to create tiny chambers and fluidic networks in quartz, silica, glass, or polymeric chips. Another advantage is that the fabrication techniques used to construct microfluidic devices are very amenable both to highly elaborate, multiplexed devices and also to mass production. Polymeric or plastic microfluidic devices have the additional advantage of being relatively inexpensive to manufacture. In a manner similar to that for microelectronics, microfluidic technologies enable the fabrication of highly integrated devices for performing several different functions on the same substrate chip.
Microfluidic devices can direct the flow of liquid chemical reagents similar to the way semiconductors direct the flow of electrons. Reagents can be diluted, mixed, or reacted with other reagents prior to analysis by capillary electrophoresis or electrochromatography—all on a single chip. As such, microfluidic devices can be designed to accommodate virtually any analytic biochemical process. Plastic, or polymeric, microfluidic devices are particularly attractive because of the low cost and relative ease of manufacture compared to glass devices. However, laser-induced fluorescence detection in polymeric microchips presents some unique challenges. Because a plastic substrate (in which the microchannels are formed) is substantially more fluorescent than freestanding silica capillaries, spatial selection is required to isolate the fluorescent signal originating from within the microchannel from fluorescence originating in the substrate material. In the past, this has typically been achieved with a confocal system; measurement of multiple channels then requires mechanical scanning of the optical elements. Examples of two different conventional laser-induced fluorescence detection systems are shown in FIGS. 1(a) and (b).
FIG. 1(a) shows a conventional confocal arrangement from Leica Microsystems (http://www.confocal-microscopy.com/website/sc_llt.nsf). In the confocal microscope shown all structures out of focus are suppressed at image formation. This is obtained by an arrangement of diaphragms, which, at optically conjugated points of the path of rays, act as a point light source and as a point detector respectively. Rays from out-of-focus areas are suppressed by the detection pinhole. The depth of the focal plane is, besides the wavelength of light, determined in particular by the numerical aperture of the objective used and the diameter of the diaphragm. With a wider detection pinhole the confocal effect can be reduced. To obtain a full image, the image point is moved across the specimen by mirror scanners. The emitted/reflected light passing through the detector pinhole is transformed into electrical signals by a photomultiplier and displayed on a computer monitor screen.
Typically, confocal arrangements, such as that shown in FIG. 1(a), would need to be augmented with additional optics behind the pinhole to direct light onto the detector(s), especially if multiple spectral bands were being examined. Thus, such a system is complex and includes moving parts.
FIG. 1(b) shows a conventional ball lens—optical coupling arrangement, with a 2 mm diameter ball lens 110 and a 1 mm core diameter fiber 60, and the ball lens 110 positioned to collimate light from source S. One advantage of a ball lens system over a confocal system is simplicity in assembly and alignment, as well as compactness. However, as can be seen in FIG. 1(b), the working distance d2 is very short, thus making this arrangement unsuitable in many situations. Further, if a conventional ball lens system were used in an epi-illumination setup for measurements on a plastic microfluidic device, the background fluorescence from the substrate would severely limit the sensitivity.
In sum, while plastic microchips remain attractive because of the low cost and relative ease of manufacture compared to glass systems, many drawbacks exist in using plastic microchips with conventional laser-induced fluorescence detection systems.
Accordingly, modern technology requires new detection systems that are simple and inexpensive to construct and operate, particularly when performing multiplexed measurements, in microchips with multiple channels.